Plant genetic engineering technology has made significant progress during the last 10 years. It has become possible to introduce stably foreign genes into plants. This has provided exciting opportunities for modern agriculture. Derivatives of the Ti-plasmid of the plant pathogen, Agrobacterium tumefaciens, have proven to be efficient and highly versatile vehicles for the introduction of foreign genes into plants and plant cells. In addition, a variety of free DNA delivery methods, such as electroporation, microinjection, pollen-mediated gene transfer and particle gun technology, have been developed for the same purpose.
The major aim of plant transformations by genetic engineering has been crop improvement. In an initial phase, research has been focussed on the engineering into plants of useful traits such as insect-resistance. In this respect, progress in engineering insect resistance in transgenic plants has been obtained through the use of genes, encoding ICPs, from Bt strains (Vaeck et al, 1987). A Bt strain is a spore forming gram-positive bacterium that produces a parasporal crystal which is composed of crystal proteins which are specifically toxic against insect larvae. Bt ICPs possess a specific insecticidal spectrum and display no toxicity towards other animals and humans (Gasser and Fraley, 1989). Therefore, the Bt ICP genes are highly suited for plant engineering purposes.
For more than 20 years, Bt crystal spore preparations have been used as biological insecticides. The commercial use of Bt sprays has however been limited by high production costs and the instability of crystal proteins When exposed in the field (Vaeck et al, 1987). The heterogeneity of Bt strains has been well documented. Strains active against Lepidoptera (Dulmage et al, 1981), Diptera (Goldberg and Margalit, 1977) and Coleoptera (Krieg et al, 1983) have been described.
Bt strains produce endogenous crystals upon sporulation. Upon ingestion by insect larvae, the crystals are solubilzed in the alkaline environment of the insect midgut giving rise to a protoxin which is subsequently proteolytically converted into a toxic core fragment or toxin of 60-70 Kda. The toxin causes cytolysis of the epithelial midgut cells. The specificity of Bt ICPs can be determined by their interaction with high-affinity binding sites present on insects' midgut epithelia.
The identification of Bt ICPs and the cloning and sequencing of Bt ICP genes has been reviewed by Hofte and Whiteley (1989). The Bt ICP genes share a number of common properties. They generally encode insecticidal proteins of 130 kDa to 140 kDa or of about 70 kDa, which contain toxic fragments of 60.+-.10 kDa (Hofte and Whiteley, 1989). The Bt ICP genes have been classified into four major groups according to both their structural similarities and insecticidal spectra (Hofte and Whiteley, 1989): Lepidoptera-specific (CryI), Lepidoptera- and Diptera-specific (CryII), Coleoptera-specific (CryIII) and Diptera-specific (CryIV) genes. The Lepidoptera-specific genes (CryI) all encode 130-140 kDa proteins. These proteins are generally synthesized as protoxins. The toxic domain is localized in the N-terminal half of the protoxin. Deletion analysis of several CryI genes confirm that 3' portions of the protoxins are not absolutely required for toxic activity (Schnepf et al, 1985). CryII genes encode 65 kDa proteins (Widner and Whiteley, 1985). The CryIIA proteins are toxic against both Lepidoptera and Diptera while the CryIIB proteins are toxic only to Lepidopteran insects. The Coleoptera-specific genes (CryIII) generally encode proteins with a molecular weight of about 70 kDa (Hofte and Whiteley, 1989). The CryIIIA gene expressed in E. coli directs the synthesis of a 72 kDa protein which is toxic for the Colorado potato beetle. This 72 kDa protein is processed to a 66 kDa protein by spore-associated bacterial proteases which remove the first 57 N-terminal amino acids (McPherson et al, 1988). Deletion analysis demonstrated that this type of gene cannot be truncated at its 3'-end without the loss of toxic activity (Hofte and Whiteley, 1989). Recently an anti-coleopteran strain, which produces a 130 kDa protein, has also been described (European patent application ("EPA") 89400428.2). The CryIV class of crystal protein genes is composed of a heterogeneous group of Diptera-specific crystal protein genes (Hofte and Whiteley, 1989).
The feasibility of generating insect-resistant transgenic crops by using Bt ICPs has been demonstrated (Vaeck et al, 1987; Fischhoff et al, 1987 and Barton et al, 1987). Transgenic plants offer an attractive alternative and provide an entirely new approach to insect control in agriculture which is at the same time safe, environmentally attractive and cost-effective (Meeusen and Warren, 1989). Successful insect control has been observed under field conditions (Delannay et al, 1989; Meeusen and Warren, 1989).
In all cases, Agrobacterium-mediated gene transfer has been used to express chimeric Bt ICP genes in plants (Vaeck et al, 1987; Barton et al, 1987; Fischhoff et al, 1987). Bt ICP genes were placed under the control of a strong promoter capable of directing gene expression in plant cells. It is however remarkable that expression levels in plant cells were high enough only to obtain insect-killing levels of Bt ICP genes when truncated genes were used (Vaeck et al, 1987; Barton et al, 1987). None of the transgenic plants containing a full-length Bt ICP gene produced insect-killing activity. Moreover, Barton et al. (1987) showed that tobacco calli transformed with the entire Bt ICP coding region became necrotic and died. These results indicate that the Bt ICP gene presents unusual problems that must be overcome to obtain significant levels of expression in plants. Even when using a truncated Bt ICP gene for plant transformation, the steady state levels of Bt ICP mRNA obtained in transgenic plants are very low relative to levels produced by both an adjacent NPT II-gene, used as a marker, and by other chimeric genes (Barton et al, 1987; Vaeck et al, 1987). Moreover, the Bt ICP full size mRNA cannot be detected by Northern blot analysis. Similar observations were made by Fischhoff et al. (1987); they reported that the level of Bt ICP mRNA was much lower than expected for a chimeric gene expressed from the CaMV35S promoter. In other words, the cytoplasmic accumulation of the Bt mRNA, and consequently the expression of the Bt ICP protein in plant cells, are extremely inefficient. By contrast, in microorganisms, it has been shown that truncated Bt ICP genes are less favorable than full-length genes (Adang et al, 1985), indicating that the inefficient expression is solely related to the heterologous expression of Bt ICP genes in plants.
The problem of obtaining significant Bt ICP expression levels in plant cells seems to be inherent and intrinsic to the, wild-type Bt ICP genes. Furthermore, the relatively low and poor expression levels obtained in plants appears to be a common phenomenon for all Bt ICP genes.
It is known that there are six steps at which gene expression can be controlled neucaryotes (Darnell, 1982):
1) Transcriptional control PA1 2) RNA processing control PA1 3) RNA transport control PA1 4) mRNA degradation control PA1 5) translational control PA1 6) protein activity control PA1 inactivating, preferably destroying or removing, in the coding region of the gene, at least one of the following process-directing sequence elements: PA1 the process-directing sequence elements preferably being inactivated by introducing translationally neutral modifications in the coding region of the gene by: PA1 1) the coding region of the modified gene; PA1 2) a promoter suitable for directing transcription of the modified gene in the plant cells; and PA1 3) suitable transcript 3' end formation and polyadenylation signals for expressing the modified gene in the plant cells. PA1 a cell of a plant, the nuclear genome of which has been transformed to contain, preferably stably integrated therein, the modified gene particularly the chimeric gene; PA1 cell cultures consisting of the plant cell; PA1 a plant which is regenerated from the transformed plant cell or is produced from the so-regenerated plant, the genome of which contains the modified gene, particularly the chimeric gene, and which shows improved properties, e.g., resistance to insect pests; PA1 seeds of the plant; and PA1 a vector for stably transforming the nuclear genome of plant cells with the modified gene, particularly the chimeric gene.
For all genes, transcriptional control is considered to be of paramount importance (The Molecular Biology of the Cell, 1989).
In European patent publications ("EP") 385,962 and 359,472 efforts to modify the codon usage of Bt ICP genes to improve their expressions in plant cells have been reported. However, wholesale (i.e., non-selective) changes in codon usage can introduce cryptic regulatory signals in a gene, thereby causing problems in one or more of the six steps mentioned above for gene expression, and thus inhibiting or interfering with transcription and/or translation of the modified foreign gene in plant cells. For example, changes in codon usage can cause differential rates of mRNA production, producing instability in the mRNA, so produced (e.g., by exposure of regions of the mRNA, unprotected by ribosomes, to attack and degradation by cytoplasmic enzymes). Changes in codon usage also can inadvertantly interfere with expression of the so-modified gene. EP 359,472 suggests that plant consensus splice sites, if present in the native Bt tenebrionis coding region, be modified so as to eliminate potentially deleterious sequences.
PCT patent publication WO 91/16432 describes the slow rate and/or level of nuclear production of a Bt ICP mRNA as an important cause of the relatively low expression levels of Bt ICP genes in plant cells. In particular, RNA polymerase elongation was found to be hindered in a specific region of the genes.